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Oral fluid rapid assay for hepatitis c virus (hcv) antibodies using non-antibody labeling of lga molecules recognizing hcv peptide epitopes

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Oral fluid rapid assay for hepatitis c virus (hcv) antibodies using non-antibody labeling of lga molecules recognizing hcv peptide epitopes

A method and device to detect Hepatitis C (HCV) antibodies in oral fluid is provided. This method introduces a non-antibody detection molecule that labels all classes of patient antibodies in oral fluid, followed by the specific concentration of labeled anti-HCV antibodies by selective capture in a trapping zone consisting of peptide antigens derived from the HCV genome. Signal generated by the labeled antibodies present in the trapping zone is proportional to the number of anti-HCV antibodies bound to the antigens present in the trapping zone. Presence of signal derived from the capture of antibody/detection molecule complexes in the trapping zone is indicative of past exposure to HCV.

Inventors: Jonathan Zmuda, Lance A. Liotta, Gordon Whiteley
USPTO Applicaton #: #20120276521 - Class: 435 5 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Virus Or Bacteriophage

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The Patent Description & Claims data below is from USPTO Patent Application 20120276521, Oral fluid rapid assay for hepatitis c virus (hcv) antibodies using non-antibody labeling of lga molecules recognizing hcv peptide epitopes.

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This application is a continuation of U.S. patent application Ser. No. 10/912,405, filed Aug. 5, 2004, which was a continuation-in-part of U.S. patent application Ser. No. 09/938,131, filed on Aug. 23, 2001, which claimed the benefit of U.S. Provisional Patent Application Ser. No. 60/227,254, filed Aug. 23, 2000.


The ability to detect anti-HCV in oral fluid is useful for the rapid detection of HCV exposure by non-invasive means. The methods provided in the invention are also useful in the early detection of HCV infection by recognition of anti-HCV of the IgA class, monitoring of antiviral therapy, genotyping of HCV virus, determining immune response to individual HCV epitopes, and monitoring potential vaccination programs.


Hepatitis C(HCV) is the major cause of parenterally transmitted non-A, non-B hepatitis (Choo et al., 1989 Science 244:359-362; Kuo et al., 1989, Science 244:362-364) with a prevalence of 1-3% throughout the world (Davis et al., 1998, Hepatology 28(Suppl 4, pt 2):99A). Chronic disease develops in 60-85% of patients, with cirrhosis representing a major hallmark of HCV infection. Among patients whose infection progresses to cirrhosis, as many as 1-4% develop hepatocellular carcinomas annually (Fattovich et al., 1997, Gastroenterology 112:463-472). It is estimated that the need for hepatic transplantation for infected individuals will increase 5-7 fold in the next 20 years unless more effective treatments and preventative programs are introduced (Davis et al., 1998, Hepatology 28 (Suppl 4, pt 2):99A).

While additional anti-viral therapies are needed to combat the spread of HCV, equally necessary is the development of a rapid, highly sensitive and cost-effective test to detect and monitor HCV within the population. Current PCR and ELISA-based assays for the detection of HCV are costly, relatively slow and reliant upon serum or plasma as the sample fluid. The substitution of oral fluid for serum in HCV assays would provide a cost-effective, non-invasive means to conduct routine screening and would facilitate sample procurement from patient groups where serum collection is difficult, such as intravenous drug users, who constitute a significant portion of total HCV cases.

A number of oral fluid-based assays have been designed for the detection of viral antibodies with good results. Virus-specific antibodies have been detected in the oral fluid of patients infected with human immunodeficiency virus (Major et al., 1991, J. Infect. Dis. 163:699-702), hepatitis A (Stuart et al., 1992, Epdiem. Infect. 109:161-166), hepatitis B (Ben Aryeh et al., 1985, Arch. Oral Biol. 30:97-99), rubella (Saleh, 1991, J. Egypt Public Health Assoc. 66:123-124) and following immunization against polio (Zaman et al., 1991, Acta Paediatrica Scan. 80: 1166-1173), rotavirus (Ward et al., 1992, J. Med. Virol. 36: 222-225) and hepatitis A (Laufer et al., 1995, Clin. Infect. Dis. 20:868-871). For HCV, ELISA-based assays developed initially for use with serum or plasma have been modified to detect anti-HCV antibodies in oral fluid (Cameron et al., 1999, J. Viral Hepatitis 6:141-144; Elsana et al., 1998, J. Med. Virol 55:24-27; McIntyre et al., 1996, Eur. J. Clin. Microbiol. Infect. Dis. 15:882-884; Sherman et al., 1994, Amer. J. Gastroent 89:2025-2027; Thieme et al., 1992, J. Clin. Microbiol. 30:1076-1079); using a modified protocol with the HCV 3.0 ELISA (Ortho Diagnostic Systems), (McIntyre et al. 1996, Eur. J. Clin. Microbiol. Infect. Dis. 15:882-884) detected anti-HCV antibodies within a group of 18 HCV(+) and 49 HCV(−) oral fluid samples with 72% sensitivity and 98% specificity. In the same study, 100% sensitivity and 100% specificity was achieved using the Monolisa HCV assay (Sanofi Pasteur Diagnostics, France). It is unclear what the differences were that lead to the increased sensitivity of the Monolisa test, and thus care must be taken in the interpretation of results obtained from tests not designed specifically for use with oral fluid. None of these assays has achieved the sensitivity required for a rapid point of care test. None of these assays has disclosed the special role of oral fluid IgA in human oral fluid as a key determinant of sensitivity and specificity for HCV screening.

An intrinsic difficulty in designing oral fluid-based diagnostic assays, however, is detecting a sufficient proportion of the relatively low levels of antibody present in oral fluid to generate a meaningful diagnostic result. Indeed, it is estimated that overall antibody levels are 800-1000-fold lower in oral fluid than in serum (Parry et al., 1987, Lancet 2:72-75) making detection sensitivity of the utmost importance in oral fluid-based tests. While this problem is significant, an HCV assay designed to be used specifically with oral fluid as the diagnostic fluid, and not simply a serum-based assay modified for use with saliva, could overcome this complication and provide an important test for HCV in the population.



The invention disclosed is a means to detect antibodies against HCV using oral fluid as a sample medium. Assays in the prior art have not achieved the sensitivity and specificity required to rapidly screen HCV infection in human oral fluid. Most critically, the use of a labeled detection molecule that recognizes not only IgG, but all classes of immunoglobulins, enhances the ability to detect anti-HCV in oral fluid in an ELISA format or using a flow-through system. When detecting anti-HCV using a labeled detection molecule that recognizes only anti-HCV of the IgG class, detection sensitivity was vastly reduced. The incorporation of a detection method that labels multiple classes of anti-HCV, on the other hand, allows for increased detection sensitivity of samples that would otherwise be scored negative using a detection method that only recognizes IgG.

By coupling this detection method to an assay that utilizes a membrane with immobilized HCV peptide antigens present as a trapping zone, followed by subsequent flow of sample through the trapping zones and selective binding of labeled antibodies specific for HCV epitopes within the trapping zone, an immunoassay for the detection of anti-HCV can be performed in a short time period (<15 minutes). The ability to use oral fluid as a sample is of great value to such a rapid diagnostic tool since oral fluid can be collected rapidly and used immediately following collection. An assay using oral fluid, performed on a miniature test platform, analyzed in a small light gathering machine, and able to be completed within 15 minutes from start to finish would be of enormous value as a screening agent for HCV in the population. By decreasing the time of the assay and eliminating the need for invasive blood-based sample acquisition, such an assay would certainly increase the ability to screen, detect and monitor HCV within the population.

The use of an assay to detect anti-HCV in saliva would also be of benefit in the rapid and non-invasive detection of antibodies following vaccinations and monitoring of vaccination efficacy over time, monitoring therapeutic response of patients to treatment regimes and screening for early infection, as IgA antibodies are known to be an important part of the early stages of the immune response.

Thus, the present invention seeks to overcome the deficiencies of the prior technology by designing an HCV assay that would meet the following objectives. A first objective is that the test is non-invasive, generates minimal risk of infection to those administering the test and can be performed from start to finish by non-medical personnel.

A second objective is that the test is rapid (<15 min.).

A third objective is that the test is specialized to detect the specific class of anti-HCV antibodies in oral fluid, and is not simply a modification of a current serum-based assay.

A fourth objective is that the test incorporates a number of different HCV antigens to minimize false negative results.

A fifth objective is that the test is adaptable to future incarnations of the assay to meet specific diagnostic needs, and that it is sensitive enough to detect extremely low levels of anti-HCV.


FIG. 1. Characterization of multiple classes of anti-HCV present in serum and oral fluid. Paired serum/oral fluid samples were screened by HCV 3.0 ELISA using enzyme-conjugated antibodies specific for human IgG, IgM or IgA, respectively. (A) In serum, high levels of anti-HCV IgG and IgM class antibodies are detectable, while relatively little anti-HCV IgA is present. (B) In oral fluid, the majority of antibodies detectable are of the IgG or IgA class with little or no anti-HCV IgM present.

FIG. 2: Components of the HCV strip immunoassay. (A) Top view of disassembled assay cassette showing the position of the nitrocellulose test strip as well as the top and bottom wicks and the substrate-coated gelbond. The “trapping zone” is located directly beneath the substrate-coated gelbond. The trapping zone and substrate-coated gelbond are kept from contacting one another until such time as the cassette is inserted into the luminometer for reading. (B) Top view of an assembled cassette with the conjugate hinge in the open position. (C) Top view of an assembled cassette with the conjugate hinge in the closed position. Also visible in C is the chase injection port and the luminescence measuring window. (D) Side view of assembled cassette showing the conjugate hinge in the open position as well as the lever on the back of the cassette that is contacted by the Junior luminometer upon insertion to bring the anti-HCV/anti-human-AP complex captured in the trapping zone into contact with the substrate-coated gelbond suspended above and thus initiate the luminescence reaction.

FIG. 3: Dose response curve for spiked monoclonal anti-HCV antibodies in an HCV(−) oral fluid sample. Monoclonal anti-HCV antibodies were spiked into an HCV(−) oral fluid sample to test the ability of the mixed antigen trap to capture anti-HCV antibodies. (A) Dose response curve for spiked monoclonal anti-HCV antibodies present in oral fluid at concentrations ranging from 0-117 μg/ml. (B) Digital photograph of nitrocellulose test strips post-stained with NBT/BCIPT corresponding to the data points in (A). Staining within coherent trapping zones is visible in all spiked samples while no staining is present in the non-spiked control.

FIG. 4. Screening of 64 known HCV(+) saliva samples on strip immunoassay. A cutoff was determined using the mean of 14 HCV(−) saliva samples+2SD. 63/64 known HCV(+) saliva samples generated values above the calculated cutoff for a sensitivity of 98.4%.

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